The capability of the high speed countercurrent chromatograph was evaluated in separation of polar dipeptides with a new set of multilayer coils of a small helical diameter. Using a hydrophilic two-phase solvent system composed of n-butanol, acetic acid and water at 4:1:5 volume ratio, four components (L-tyrosylglycine, L-valyl-L-tyrosine, L-leucyl-L-tyrosine and L-tryptophyl-L-tyrosine) were separated at a flow rate of 1 to 2 ml/min. Best results were obtained by eluting the upper nonaqueous phase in a head to tail direction under 800 rpm of coil rotation. Four components were completely resolved in 5 to 10 hours.